Neutrophils are polymorphonuclear leukocytes (PMNs) of the blood that play a major role in almost all forms of acute inflammation and many forms of chronic inflammation, such as rheumatoid arthritis. When triggered, neutrophils secrete potent oxidants and proteases that contribute to inflammation but also help protect the host from infection. Neutrophils respond to soluble inflammatory mediators by migrating to the site of tissue injury and by ingesting and destroying invading pathogens and damaged tissue, leading, ultimately, to resolution and tissue repair. An inherent dilemma of anti-inflammatory therapy, the risk of impairing host defense, is particularly problematic with neutrophils.
To participate effectively in the inflammatory process, neutrophils must leave the bloodstream and migrate into the tissues. The initial step in this process is adherence to the vascular endothelium. Adherence of neutrophils to endothelium, their diapedesis from venules into tissues, and their release of peptides, proteases, and reactive oxygen intermediates underlie both their killing of bacteria and their damage to tissues.
Full activation of neutrophils by soluble host products requires a binary signal. One part of the signal consists of integrin ligation during adherence to extracellular matrix. Simultaneously, a factor such as tumor necrosis factor (TNF), lymphotoxin, C5a, formylated peptides, macrophage inflammatory protein (MIP-1), granulocyte-specific colony stimulating factor (G-CSF), or granulocyte-macrophage-specific colony stimulating factor (GM-CSF), must engage its receptor(s) on the neutrophil. Klebanoff et al., “Stimulation of Neutrophils by Tumor Necrosis Factor,” J. Immunol. 136:4220-4225 (1986); Nathan, “Neutrophil Activation on Biological Surfaces. Massive Secretion of Hydrogen Peroxide in Response to Products of Macrophages and Lymphocytes,” J. Clin. Invest. 80:1550-1560 (1987); Wolpe et al., “Macrophages Secrete a Novel Heparin-Binding Protein with Inflammatory and Neutrophil Chemokinetic Properties,” J. Exp. Med. 167:570-581 (1988); Nathan, “Respiratory Burst in Adherent Human Neutrophils: Triggering by Colony-Stimulating Factors CSF-GM and CSF-G,” Blood 73:301-306 (1989). TNF has been studied extensively with respect to both the mechanisms by which it activates adherent neutrophils (Nathan C. F., “Neutrophil Activation on Biological Surfaces. Massive Secretion of Hydrogen Peroxide in Response to Products of Macrophages and Lymphocytes,” J. Clin. Invest. 80:1550-1560 (1987); De La Harpe et al., “Adenosine Regulates the Respiratory Burst of Cytokine-Triggered Human Neutrophils Adherent to Biologic Surfaces,” J. Immunol. 143:596-602 (1989); Laudanna et al., “Tumor Necrosis Factor-Alpha/Cachectin Activates the O2−-Generating System of Human Neutrophils Independently of the Hydrolysis of Phosphoinositides and the Release of Arachidonic Acid,” Biochem. Biophys. Res. Commun. 166:308-315 (1990); Nathan et al., “Tumor Necrosis Factor and CD11/CDI8 (beta 2) Integrins Act Synergistically to Lower cAMP in Human Neutrophils,” J. Cell Biol. 111:2171-2181 (1990); Dri et al., “Effect of Biological Surfaces on Neutrophil O2-Production and its Relationship to the CD11b/CDI8 Integrin-Dependent Adherence, Int. J. Tissue React. 13:193-201 (1991); Dapino et al., “Induction of Neutrophil Respiratory Burst by Tumour Necrosis Factor-Alpha; Priming Effect of Solid-Phase Fibronectin and Intervention of CD11b-CD18 Integrins,” Clin. Exp. Immunol 94:533-538 (1993); Fuortes et al., “Adhesion-Dependent Protein Tyrosine Phosphorylation in Neutrophils Treated with Tumor Necrosis Factor,” J. Cell Biol. 120:777-784 (1993); Nathan et al., “Albumin inhibits Neutrophil Spreading and Hydrogen Peroxide Release by Blocking the Shedding of CD43 (Sialophorin, Leukosialin),” J. Cell Biol. 122:243-256 (1993); Liles et al., “Cross-Linking of CDI8 Primes Human Neutrophils for Activation of the Respiratory Burst in Response to Specific Stimuli: Implications for Adhesion-Dependent Physiological Responses in Neutrophils,” J. Leukoc. Biol. 58:690-697 (1995); Richter et al., “TNF-Induced Superoxide Anion Production in Adherent Human Neutrophils Involves Both the p55 and p75 TNF Receptor,” J. Immunol. 154:4142-4149 (1995); Puortes et al., “Ceramide Selectively Inhibits Early Events in the Response of Human Neutrophils to Tumor Necrosis Factor,” J. Leukoc. Biol. 59:451-460 (1996); Lowell et al., “Deficiency of Src Family Kinases p59/61hck and p58c-fgr Results in Defective Adhesion-Dependent Neutrophil Functions,” J. Cell Biol. 133:895-910 (1996)) and the benefit of its neutralization in inflammatory disorders such as rheumatoid arthritis (Feldmann, “Development of Anti-TNF Therapy for Rheumatoid Arthritis,” Nat. Rev. Immunol. 2:364-371 (2002)); ankylosing spondylitis (Braun et al., “Treatment of Active Ankylosing Spondylitis with Infliximab: A Randomised Controlled Multicentre Trial,” Lancet. 359:1187-1193 (2002)); and Crohn's Disease (Sandborn et al., “Antitumor Necrosis Factor Therapy for Inflammatory Bowel Disease: A Review of Agents, Pharmacology, Clinical Results, and Safety,” Inflamm. Bowel Dis. 5:119-133 (1999)).
Following adherence, the immune response of neutrophils involves chemotaxis, phagocytosis, and degranulation. Neutrophils sense and respond to gradients of activating agents (chemokines), which stimulate the neutrophil to move towards the gradient in a process known as chemotaxis (Brown S S., “Structure and Function of Profilin,” Cell Motil Cytoskel 17:71-75 (1990); Southwick et al., “Contractile Proteins in Leukocyte Function,” Semin Hematol 30:305-310(1984)). Activated neutrophils are also capable of phagocytosing, i.e., engulfing foreign or damaged material, an important aspect of the inflammatory response. To engulf a particle, neutrophils extend pseudopodia, which engulf the offending material, trapping the material inside the cell in a compartment known is a phagosome (Wright S. D., “Receptors for Complement and the Biology of Phagocytosis,” In Inflammation 2nd ed. 477-496 Raven Press New York (1992)). Cytoplasmic-bound granules, the primary and secondary granules of the neutrophil, which contain a multitude of effectors proteins, fuse with the phagosome, placing effector proteins in direct contact with the ingested material. Components of the primary granules include lysozyme, which can digest the peptidoglycan component of most bacterial cell walls, and elastase, cathepsin G, defensins, bacterial permeability-increasing protein (BPI), and myeloperoxidase, which converts hydrogen peroxide generated by NADPH oxidase and hydrochloric acid to hypochlorous acid, all with inherent antibacterial activity. Among the proteins contained in the secondary granules are lactoferrin, an iron-binding protein with some antibacterial activity. The secondary granules also contain stored sources of CR3 and other receptors for neutrophil activation agents, as well as stored membrane components of NADPH oxidase. NADPH oxidase is a crucial component of the neutrophil host defense mechanism. This enzyme assembles on the phagosomal membrane to generate superoxide anion from molecular oxygen and free electrons. Superoxide is then converted to the toxic metabolite hydrogen peroxide by the actions of superoxide dismutase, or to hypochlorous acid by the primary granule component myeloperoxidase (DeLeo et al., “Assembly of Phagocyte NADPH Oxidase: Molecular Interaction of Oxidase Proteins,” J. Leukocyte Biol 60:677-691 (1996); Leusen et al., “Interactions Between the Components of the Human NADPH Oxidases: Intrigues in the Phox Family,” J. Clin Lab Med 128: 461-476 (1996); Wientjes et al., NADPH Oxidase and the Respiratory Burst,” Semin Cell Biol 6:357-365 (1995); Henderson et al., “NADPH Oxidases of Neutrophils,” Biochim Biophys Acta 1273:87-107 (1996)). This ability to generate toxic oxygen metabolites is crucial to host defense against microbes.
The histotoxic impact of neutrophils is prominent in several inflammatory settings that are not thought to involve bacterial infection, such as rheumatoid arthritis, Crohn's Disease, and ischemia-perfusion syndrome, or in which neutrophil-mediated injury can occur at sites remote from invading bacteria, as in the acute respiratory distress and systemic inflammatory response syndromes. The stimuli that activate neutrophils in these settings are host-derived mediators (e.g., TNF), rather than bacteria themselves. What is needed is a method for inhibiting some inflammatory functions of activated neutrophils while sparing antimicrobial functions. Such a method would be particularly useful for preventing and treating inflammatory disorders related to respiratory burst in neutrophils mediated by effector proteins such as TNF.
The present invention is directed to overcoming these and other deficiencies in the art.